The human immune response to Mycobacterium tuberculosis (Mtb) permits establishment of latency and in some individuals, subsequent reactivation and disease. The host-pathogen interactions that prevent sterilizing immunity are not well understood. Emerging research demonstrates that among Mtb-nave hosts, Mtb-reactive innate T cells with effector functions associated with protective immunity are present. Moreover, human nave T cells are capable of using Toll-like receptors (TLR), molecules typically associated with innate immunity, to directly receive co-stimulatory signals from Mtb ligands. We hypothesize that Mtb-reactive innate T cells, and nave T cells with functional innate co-stimulatory receptors, are a source of pro-inflammatory mediators such as IFN-? during the initial stages of Mtb infection. This early production of IFN-? and other effector molecules may augment antimicrobial responses by Mtb-infected macrophages, as well as IL-12 production by Mtb- infected dendritic cells. Defining and characterizing the repertoire of Mtb-reactive innate T cells, and nave T cells with functional TLR, is an essential first step in the development of vaccines and immune-based therapeutics that expand these cells in order to bolster sterilizing immune responses against Mtb. The objectives of this proposal are to characterize the phenotype, frequency, effector potential, and gene expression profile of Mtb-reactive, innate T cells, as well as nave T cell subsets (including regulatory T cells), that utilize TLR to respond o Mtb ligands, among Mtb-nave hosts from birth to adulthood. The specific aims of this proposal are focused on investigating innate T cell responses to Mtb among antigen-inexperienced neonates and Mtb-naive infants and adults. We will use a novel assay to identify and quantify non-classically restricted Mtb-reactive T cells among donors of different ages, and a combination of flow cytometry, multiplex cytokine arrays, inorganic mass spectrometry, and gene expression profiling to delineate their phenotypes, functional capabilities, and molecular profiles. We will characterize the phenotype, functional capabilities, gene expression profiles, and T cell receptor diversity of nave T cell subsets responsive to TLR-mediated co-stimulation among neonates, infants, and adults. We will also determine if age impacts TLR-mediated alterations of the suppressive functions of regulatory T cells. Our approach will allow us to determine if TLR-responsive nave T cells isolated from antigen-inexperienced hosts, such as newborns, represent a unique subset of innate T cells. Understanding the ontogeny of innate T cells and the capacity of T cells to utilize innate receptors to alter their activation and functio, will advance our understanding of the full potential of T cell developmental pathways, as well as their contribution to host immune defense. In addition, as neonates and infants are exquisitely vulnerable to severe disease phenotypes from infectious pathogens, defining the innate capacity of their T cells to respond to a pathogen such as Mtb, can be used to inform the development of next generation vaccines and therapeutics utilized in infancy.